1. Field of the Invention
This invention relates generally to the field of regulation of gene expression and specifically to a novel protein, I.kappa.B-.beta., which contributes to the regulation of the transcription factor, NF-.kappa.B.
2. Description of Related Art
The nuclear factor-kappa B (NF-.kappa.B) is an inducible transcription factor which participates in the regulation of multiple cellular genes after treatment of cells with factors such as phorbol ester, lipopolysaccharide (LPS), interleukin-1 (IL-1) and tumor necrosis factor-.alpha. (TNF-.alpha.). These genes are involved in the immediate early processes of immune, acute phase, and inflammatory responses. NF-.kappa.B has also been implicated in the transcriptional activation of several viruses, most notably the type 1 human immunodeficiency virus (HIV-1) and cytomegalovirus (CMV) (Nabel, et al., Nature, 326:711, 1987; Kaufman, et al., Mol. Cell. Biol., 7:3759, 1987; Sambucetti, et al., EMBO J, 8:4251, 1989).
NF-.kappa.B is a dimeric transcription factor that binds and regulates gene expression through decameric cis-acting .kappa.B DNA motifs. Although a p50/p65 heterodimer has traditionally been referred to as NF-.kappa.B and remains the prototypical and most abundant form, it has been recognized recently that several distinct but closely related homo- and heterodimeric factors are responsible for .kappa.B site-dependent DNA binding activity and regulation. The various dimeric factors are composed of members of the family of Rel-related polypeptides. One subclass of this family, distinguished by its proteolytic processing from precursor forms and lack of recognized activation domains, includes p50 (NFKB1) and p50B (NFKB2, p52), whereas the second subclass contains recognized activation domains and includes p65 (RelA), RelB, c-Rel, and the Drosophila protein Dorsal. All Rel-related members share a 300-amino acid region of homology, RHD, responsible for DNA binding and dimerization, called the Rel homology domain. In the cytoplasm, NF-.kappa.B and Rel proteins form a "Rel complex".
Activation of the NF-.kappa.B transcription factor and various related forms can be initiated by a variety of agents, including TNF.alpha., phorbol 12-myristate 13-acetate (PMA), interleukin-1 (IL-1) and interleukin-2 (IL-2). Activation proceeds through a post-translational event in which preformed cytoplasmic NF-.kappa.B in the Rel complex is released from a cytoplasmic inhibitory protein. A common feature of the regulation of transcription factors which belong to the Rel-family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as I.kappa.Bs (Baeuerle and Baltimore, Cell, 53:211-217, 1988; Beg and Baldwin, Genes Dev. 7:2064-2070, 1993; Gilmore and Morin, Trends in Genetics, 9:427-433, 1993). Treatment of cells with different inducers, e.g., IL-1, TNF-.alpha., LPS, dsRNA or PMA, results in dissociation of the cytoplasmic complexes and translocation of free NF-.kappa.B to the nucleus (Grilli, et al., International Rev. of Cytology, 143:1-62, 1993; Baeuerle and Henkel, Annu. Rev. Immunol., 12:141-179, 1994). The dissociation of the cytoplasmic complexes is thought to be triggered by the phosphorylation and subsequent degradation of the I.kappa.B protein (Palombella, et al., Cell, 78:773-785, 1994; Ghosh and Baltimore, Nature, 344:678-682,1990). Transient I.kappa.B phosphorylation has been observed in several in vivo activation studies (Brown, et al., Proc. Natl. Acad. Sci., U.S.A., 90:2532, 1993; Beg, et al., Mol. Cell. Biol., 13:3301, 1993).
There are two major biochemically characterized forms of I.kappa.B proteins in mammalian cells, I.kappa.B-.alpha. and I.kappa.B-.beta. (Ghosh and Baltimore, supra; Zabel and Baeuerle, Cell, 61:255-265, 1990). In addition, three other proteins have been cloned or implicated as I.kappa.Bs: chicken pp40, the mammalian I.kappa.B-.alpha. homolog and inhibitor of the chicken oncogene, v-rel (Davis, et al., Science, 253:1268-1271, 1991; Stephens, et al., Proc. Natl. Acad. Sci. USA, 80:6229-6232, 1983); I.kappa.B-.gamma., a tissue specific form that arises from an alternative splice yielding the C-terminus of the p105 protein (Inoue, et al., Cell 68:1109-1120, 1992); and the candidate oncogene, Bcl-3 (Franzoao, et al., Nature, 359:339-342, 1992; Nolan, et al., Mol. Cell Biol., 13:3557-3566, 1993; Ohno, et al., Cell, 60:991-997, 1990). A common feature of all of the cloned I.kappa.B proteins is the presence of multiple copies of a sequence motif known as ankyrin repeats (Beg and Baldwin, supra; Gilmore and Morin, supra).
However, while I.kappa.B-.gamma. has been detected only in mouse pre-B cells (Ghosh, et al., 1990; Inoue, et al., supra), Bcl-3 can only be detected in very low amounts in some tissues. In addition, both I.kappa.B-.gamma. (Liou, et al., EMBO J., 11:3003-3009, 1992) and Bcl-3 (Franzoso, et al., supra, Naumann, et al., EMBO J., 12:213-222, 1993; Nolan, et al., Cell, 64:961-969, 1991; Wulczyn, et al., Nature, 358:597-599, 1992) are specific for NF.kappa.B p50 dimers and only I.kappa.B-.alpha. and I.kappa.B-.beta. interact with p65 and c-Rel, thus indicating that the responsibility for regulating the proto-typical NF-.kappa.B activity is primarily carried out by these I.kappa.B isoforms.
I.kappa.B-.alpha. was cloned previously and its regulation has been studied quite extensively (Beg, et al., Mol. Cell Bio., 13:3301-3310, 1993; Beg, et al., Genes Dev., 6:1899-1913, 1992; Brown, et al., Proc. Natl. Acad. Sci USA, 90:2532-2536, 1993; Davis, et al., supra; Haskill, et al., Cell, 65:1281-1289, 1991; Henkel, et al., Nature, 365:82-85, 1993; Mellitis, et al., Nucl. Acids Res., 21:5059-5066, 1993; Miyamoto, et al., Mol. Cell Biol., 14:3276-3282, 1994; Palombella, et al., supra; Rice and Ernst, EMBO J., 12:4685-4695, 1993; Scott, et al., Genes Dev., 7:1266-1276, 1993; and Sun, et al., Science, 259:1912-1915, 1993). These studies indicated that I.kappa.B-.alpha. regulated NF-.kappa.B activity through a novel auto-regulatory feed-back loop. Signals that led to an induction of NF-.kappa.B activity resulted in the phosphorylation and rapid loss of I.kappa.B-.alpha. protein through proteolysis. However, the induced, nuclear NF-.kappa.B caused the subsequent upregulation of I.kappa.B-.alpha. mRNA levels due to the presence of NF-.kappa.B sites in the I.kappa.B-.alpha. promoter (de Martin, et al., EMBO J.,, 12:2773-2779, 1993; Le Bail, et al., EMBO J., 12:5043-5049, 1993). The newly synthesized I.kappa.B-.alpha. mRNA was translated, and the accumulated I.kappa.B-.alpha. protein helped to shut down the NF-.kappa.B response, thus ensuring that responsive genes were activated only transiently. While this model explained some aspects of the regulation of NF-.kappa.B activity in cells, it failed to explain how some inducers, particularly bacterial lipopolysaccharide (LPS), could cause persistent long-term activation of NF-.kappa.B for as long as 36 hours. Persistent activation of NF-.kappa.B might also occur during differentiation, either in early embryonic development or in the development of B-cells or macrophages. Because a significant portion of the cytoplasmic Rel complexes are bound to I.kappa.B-.beta., it is possible that inducers like LPS or differentiation signals caused persistent NF-.kappa.B activation by affecting I.kappa.B-.beta. complexes. However, the lack of a clone for I.kappa.B-.beta. and reagents specific for the protein had prevented the determination of how and when complexes bound to I.kappa.B-.beta. were activated.
Previous attempts to isolate and purify I.kappa.B-.beta. have been unsuccessful. A study by Ghosh and Baltimore, supra, identified I.kappa.B activity as being associated with a 35 Kd protein in rabbit tissue. Zabel and Baeuerle (Cell, 61:255, 1990) then purified a complex which included p50:p65 and two forms of I.kappa.B, as determined by two distinct activities. While I.kappa.B-.alpha. was purified to homogeneity, I.kappa.B-.beta. could only be partially purified based on a peak fraction of activity. A later study purported to purify a protein having I.kappa.B activity in the range of 40-43 KD and a pI of 4.8-5.0 (Link, et al., J. Biol. Chem., 267:239, 1992). However, the fraction containing this activity was insufficient to allow reproducible peptide maps or amino terminal sequence analysis.
NF-.kappa.B gene regulation is involved in many pathological events including progression of acquired immune deficiency disease (AIDS), the acute phase response and the activation of immune and endothelial cells during toxic shock, allograft rejection, and radiation responses. In addition, NF-.kappa.B gene transactivation may be critical for HIV and CMV replication.
Therefore, identification of compositions which affect I.kappa.B-.beta./NF-.kappa.B complex integrity, and thus, affect NF-.kappa.B transactivation of genes, is critical for identification of specific inhibitors of complex dissociation, which would be effective as anti-inflammatory and immunosuppressive agents.